Sequential inactivation of rdx4 (HP0954) and frxA (HP0642) nitroreductase genes causes moderate and high-level metronidazole resistance in Helicobacter pylori

Helicobacter pylori is a human-pathogenic bacterial species that is subdivi ded geographically, with different genotypes predominating in different par ts of the world. Here we test and extend an earlier conclusion that metroni dazole (Mtz) resistance is due to mutation in rdxA (HP0954), which encodes a nitroreductase that converts Mtz from prodrug to bactericidal agent. We f ound that (i) rdxA genes PCR amplified from 50 representative Mtz(r) strain s from previously unstudied populations in Asia, South Africa, Europe, and the Americas could, in each case, transform Mtz(s) H. pylori to Mtz(r); (ii ) Mtz(r) mutant derivatives of a cultured Mtz(s) strain resulted from mutat ion in rdxA; and (iii) transformation of Mtz(s) strains with rdxA-null alle les usually resulted in moderate level Mtz resistance (16 mu g/ml). However , resistance to higher Mtz levels was common among clinical isolates, a res ult that implicates at least one additional gene. Expression in Escherichia coli of frxA (HP0642; flavin oxidoreductase), an rdxA paralog, made this n ormally resistant species Mtz(s), and frxA inactivation enhanced Mtz resist ance in rdxA-deficient cells but had little effect on the Mtz susceptibilit y of rdxA(+) cells. Strains carrying frxA-null and rdxA-null alleles could mutate to even higher resistance, a result implicating one or more addition al genes in residual Mtz susceptibility and hyperresistance. We conclude th at most Mtz resistance in H. pylori depends on rdxA inactivation, that muta tions in frxA can enhance resistance, and that genes that confer Mtz resist ance without rdxA inactivation are rare or nonexistent in H. pylori populat ions.